Design, synthesis and anti-proliferative effects in tumor cells of new combretastatin A-4 analogs

Article abstract of DOI:10.1016/j.cclet.2015.05.003

Abstract A total of 11 novel combretastatin A-4 (CA-4) analogs were designed, synthesized, and evaluated for the anti-proliferative effects in tumor cells. The compounds represent four structural classes: (i) hydrogenated derivatives, (ii) ethoxyl derivatives, (iii) amino derivatives and (iv) pro-drugs. Biological evaluations demonstrate that multiple structural features control the biological potency. Three of the compounds, sit-1, sit-2 and sit-3, have potent anti-proliferative activity against multiple cancer cell lines. Their pro-drugs were synthesized to increase water solubility. Structure-activity relationship study and Surflex-Docking were studied in this paper. These results will be useful for the design of new CA-4 analogs that are structurally related to the SAR study.

Full text of DOI:10.1016/j.cclet.2015.05.003

G Model
CCLET 3307 1–7
Chinese Chemical Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
1
2
Original article
3
4
Design, synthesis and anti-proliferative effects in tumor cells of new
Q1 combretastatin A-4 analogs
* *
Q2 Lei Zhao, Jiu-Jiu Zhou, Xin-Ying Huang, Li-Ping Cheng, Wan Pang , Zhen-Peng Kai ,
5
6
*
Fan-Hong Wu
7
School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai 201418, China
A R T I C L E I N F O
A B S T R A C T
Article history:
A total of 11 novel combretastatin A-4 (CA-4) analogs were designed, synthesized, and evaluated for the
anti-proliferative effects in tumor cells. The compounds represent four structural classes: (i)
hydrogenated derivatives, (ii) ethoxyl derivatives, (iii) amino derivatives and (iv) pro-drugs. Biological
evaluations demonstrate that multiple structural features control the biological potency. Three of the
compounds, sit-1, sit-2 and sit-3, have potent anti-proliferative activity against multiple cancer cell
lines. Their pro-drugs were synthesized to increase water solubility. Structure–activity relationship
study and Surflex-Docking were studied in this paper. These results will be useful for the design of new
CA-4 analogs that are structurally related to the SAR study.
Received 25 February 2015
Received in revised form 3 April 2015
Accepted 27 April 2015
Available online xxx
Keywords:
Combretastatin A-4
Anti-proliferative
ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Synthesized
Structure–activity relationship study
8
9
1. Introduction
sever cancer cell lines. For example, a pro-drug of CA-4, the water- 30
soluble phosphate derivative CA-4P (fosbretabulin) is now in phase 31
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Combretastatins were first extracted from the bark of the South
African bush willow tree combretum caffrum in 1982 by Pettit
et al. [1]. Those compounds may serve as a new anticancer drug
that utilized starvation tactics to attack solid tumors. Among the
combretastatin family, combretastatin A-4 (CA-4) (Fig. 1; (Z)-5-
(3,4,5-trimethoxystyryl)-2-methoxyphenol) exerts a potent inhi-
bition of tubulin polymerization by binding to the colchicine site
and, as a consequence, demonstrates strong activity in suppressing
tumor blood flow (TBF). Several studies described its ability to
induce widespread necrosis of solid tumors, including multidrug-
resistant ones, which suggested CA-4 is an attractive lead
compound for the development of novel antitubulin anticancer
agents [2,3].
However, CA-4 does not show in vivo efficacy due to its poor
pharmacokinetics resulting from its high lipophilicity, low
aqueous solubility and also isomerization of cis-double bond to
the more thermally stable trans-isomer [4–6]. So, considerable
efforts have gone into modifying CA-4 to improve its water
solubility and in vivo efficacy. To date, various CA-4 analogs have
been synthesized and reported to possess cytotoxic activity against
II clinical trials. Another combretastatin A-4 analog, the serine 32
amide AVE8062 (ombrabulin) is not only in a phase II clinical trial 33
in combination with taxanes and platinum salts in advanced solid 34
tumors, but also in a phase II/III clinical trial in patients with 35
advanced soft tissue sarcoma [7–9].
36
To study the relationship between the structure of CA-4 and the 37
anti-proliferative effect in human cancer cells (SAR study), we 38
synthesized 11 CA-4 analogs containing (i) hydrogenated deriva- 39
tives, (ii) ethoxyl derivatives, (iii) amino derivatives and (iv) pro- 40
drugs. The anti-proliferative effect of each class was tested using 41
the MTT assay in human cancer cells in vitro. To understand the 42
SAR of the CA-4 analogs, Surflex-Docking was applied to study the 43
interactions between these analogs and tubulin. Furthermore, we 44
have designed and synthesized a number of pro-drugs of potent 45
CA-4 analogs to increase the water solubility.
46
2. Experimental
2.1. Synthesis
47
48
49
2.1.1. General methods
All the chemicals and reagents were commercially available and 50
Q3
required no further purifications. Solvents (THF, DMF, CH2Cl2, 51
*
Corresponding authors.
E-mail address: pangwan@sit.edu.cn (W. Pang).
benzene) were dried and freshly distilled before use according to 52
http://dx.doi.org/10.1016/j.cclet.2015.05.003
1001-8417/ß 2015 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: L. Zhao, et al., Design, synthesis and anti-proliferative effects in tumor cells of new combretastatin A-4
analogs, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.05.003

G Model
CCLET 3307 1–7
2
L. Zhao et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
Part A
mixture was raised to 70 8C. The reaction mixture was then stirred
89
at this temperature until TLC analysis indicated the completion of
the reaction, then quenched by water, extracted with EtOAc, dried
over Na2SO4 and concentrated. Pure white 4-ethoxy-3-methox-
ybenzaldehyde 2 was obtained by crystallization (petroleum ether
(60–90 8C): EtOAc = 9:1, v/v). mp 62.1–62.2 8C. 1H NMR (CDCl3,
90
91
92
93
Part C
Part B
H3CO
H3CO
OH
OCH3
OCH3
94
Fig. 1. Structure of combretastatin A-4.
500 MHz):
d
9.84 (s, 1H), 7.41–7.45 (m, 2H), 6.96 (d, 1H, J = 5.0 Hz),
95
5.80 (s, 1H), 4.19 (q, 2H, J = 5.0 Hz), 3.93 (s, 3H), 1.50 (t, 3H,
J = 5.0 Hz).
96
97
53
54
55
56
57
58
59
60
61
62
63
64
65
66
literature procedures. Chromatographic separations were per-
formed on silica gel flash columns. TLC analyses were performed
on precoated silica gel polyester plates with a fluorescent indicator
UV 254. Melting points were determined using a melting point
apparatus (WRS-2A) and uncorrected. 1H NMR and 13C NMR
spectra were recorded on a Bruker AVANCE III at 500 MHz or on a
Bruker spectrometer at 101 Hz in chloroform-d using TMS
A
solution of 4-ethoxy-3-methoxybenzaldehyde
2
(1 g,
98
99
5.6 mmol), p-toluenesulfonicacid (60 mg, 0.31 mmol) and ethylene
glycol (6 mL, 0.1 mol) in benzene (30 mL) was refluxed for 12 h.
After cooling to room temperature, aqueous potassium carbonate
(15%, 25 mL) was added. Organic layer was washed with aqueous
potassium carbonate (15%, 50 mL), dried over Na2SO4 and
concentrated, white 2-(4-ethoxy-3-methoxyphenyl)-1,3-dioxo-
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
(
d
= 0.0 ppm) as an internal standard. IR was recorded on a
lane 3 was obtained. mp 75–77 8C. 1H NMR (CDCl3, 500 MHz):
d
NICOLET 6700 FT-IR. HRMS were recorded on a solanX 70 FT-MS
spectrometer using methanol and water (v/v = 1:1) as solvent.
LC–Mass spectra were recorded on a LCMS-2020 spectrometer
from Shimadzu Corporation with acetonitrile and water as the
mobile phase and the gradient was from 5% of acetonitrile at 0 min
to 100% of acetonitrile at 10 min.
7.21 (s, 1H), 6.83 (d, 2H, J = 5.0 Hz), 5.80 (s, 1H), 4.00 (t, 4H,
J = 5.0 Hz), 4.04 (q, 2H, J = 5.0 Hz), 3.83(s, 3H), 1.43 (t, 3H, J = 5.0 Hz).
A solution of diphenylphosphine (1 mL, 5.8 mmol) and n-butyl
lithium (2.5 mol/L in hexanes, 3 mL) in anhydrous THF (10 mL) was
stirred in an ice bath under nitrogen atmosphere. 2-(4-Ethoxy-3-
methoxyphenyl)-1,3-dioxolan 3 (1 g, 4.4 mmol) was dissolved in
anhydrous THF (5 mL) and added. Then the solution was stirred at
room temperature until TLC analysis indicated the completion of
the reaction. The mixture was quenched with water. Aqueous
phase was acidified with HCl when the yellow mixture became tea
green and the product was extracted with EtOAc, dried over
Na2SO4 and evaporated in vacuum. Pure white 4-ethoxy-3-
hydroxybenzaldehyde 5 was obtained after column chromatogra-
phy purification (petroleum ether (60–90 8C): EtOAc = 5:1, v/v).
67
68
69
70
71
72
73
2.1.2. (Z)-2-Methoxy-5-(3,4,5-trimethoxystyryl)phenol (CA-4)
Following the synthetic method from Shen et al. [10],
compound CA-4 was obtained in 35% yield. mp 116.8–117.6 8C.
1H NMR (CDCl3, 500 MHz):
d 3.69 (s, 6H), 3.84 (s, 6H), 3.85 (s, 3H),
5.58 (s, 1H), 6.41 (d, 1H, J = 12.0 Hz), 6.46 (d, 1H, J = 12.0 Hz), 6.53
(s, 2H), 6.73 (d, 1H, J = 8.5 Hz), 6.79 (dd, 1H, J = 8.5 Hz, J = 2.0 Hz),
6.92(d, 1H, J = 2.0 Hz).
mp 125–127 8C. 1H NMR (CDCl3, 500 MHz):
7.44 (m, 2H), 6.94 (d, 1H, J = 10.0 Hz), 5.80 (s, 1H), 4.22 (q, 2H,
J = 5.0 Hz), 1.50 (t, 3H, J = 5.0 Hz).
d 9.84 (s, 1H), 7.41–
74
75
76
77
78
79
80
81
82
2.1.3. 2-Methoxy-5-(3,4,5-trimethoxyphenethyl)phenol (sit-1)
A solution of compound CA-4 (1.0 g, 3.16 mmol) and palladium-
carbon (10%, 0.1 g) in MeOH (20 mL) was stirred at room
temperature for 2 h under hydrogen atmosphere. Palladium-
carbon was filtered off, the filtrate was dried over Na2SO4 and
concentrated. Compound sit-1 was obtained in 91% yield. 1H NMR
A
mixture of 4-ethoxy-3-hydroxybenzaldehyde
5
(1 g,
6.02 mmol) and potassium carbonate (1.24 g, 9 mmol) in EtOH
(20 mL) was stirred at 40 8C for 10 min. Benzyl chloride (0.7 mL,
6.1 mmol) was added through the septum. Then the reaction
mixture was refluxed for 3 h. After cooling to 50 8C, the solution
was filtered. Pure white 3-(benzyloxy)-4-ethoxybenzaldehyde 6
was obtained after the filtrate was cooled in a fridge. mp 69.8–
70.8 8C. 1H NMR (CDCl3, 500 MHz):
8H), 5.20 (s, 2H), 4.19 (q, 2H, J = 5.0 Hz), 1.50 (t, 3H, J = 5.0 Hz). 13
NMR (CDCl3, 101 MHz): 190.8, 154.7, 148.8, 136.6, 129.8, 128.5,
127.9, 127.2, 126.8, 112.3, 111.9, 71.0, 64.6, 14.6. MS (m/z): 257
(M+). ESI-HRMS (m/z): calcd. for C16H17O3 (M+H)+, 257.1178.
Found: 257.1196. IR (KBr) (
1595, 1580, 1436, 1278, 1266, 1131, 1001, 874, 802, 750.
(500 MHz, CDCl3):
d 6.81 (d, 1H, J = 3.0 Hz), 6.77 (d, 1H, J = 8.0 Hz),
6.64–6.66 (m, 1H), 6.38 (s, 2H), 5.61(s, 1H), 3.87 (s, 3H), 3.83 (s, 9H),
2.82 (s, 4H).
d 9.81 (s, 1H), 6.98–7.47 (m,
83
84
85
86
87
88
2.1.4. 5-(3-(Benzyloxy)-4-ethoxystyryl)-1,2,3-trimethoxybenzene
C
(sit-11) (Scheme 1)
d
A
mixture of 4-hydroxy-3-methoxybenzaldehyde
1
(1 g,
6.57 mmol) and potassium carbonate (1.36 g, 9.85 mmol) in
DMF (20 mL) was stirred at 40 8C for 10 min. Ethyl bromide
(0.98 mL, 13.1 mmol) was added through the septum after the
y
max, cmꢀ1): 2985, 2935, 2820, 1686,
Scheme 1. Synthesis of sit-2, sit-8, sit-11 by our group before.
Please cite this article in press as: L. Zhao, et al., Design, synthesis and anti-proliferative effects in tumor cells of new combretastatin A-4
analogs, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.05.003

G Model
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L. Zhao et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
3
137
138
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149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
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167
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169
170
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179
180
NaH (60%, 0.64 g, 26.6 mmol) was added to anhydrous DMSO
(10 mL) and the mixture was stirred at room temperature
(Scheme 2). A solution of 3,4-dihydroxybenzaldehyde 9 (1 g,
7.24 mmol) in anhydrous DMSO (5 mL) was added dropwise
through a syringe. The reaction mixture was stirred for about half
an hour. Benzyl bromine (0.86 mL, 7.3 mmol) was then added
dropwise by a syringe and the resulting solution was stirred
overnight. The mixture was neutralized with HCl (2 mol/L) and
extracted with EtOAc, dried over Na2SO4 and evaporated in
vacuum. Pure 3-(benzyloxy)-4-hydroxybenzaldehyde 10 was
obtained after column chromatography purification (petroleum
ether (60–90 8C): EtOAc = 3:1, v/v). mp 114.3–114.7 8C. 1H NMR
J = 5.0 Hz), 6.62 (d, 1H, J = 5.0 Hz), 6.38 (s, 2H), 5.63 (s, 1H), 4.09 (q, 188
2H, J = 5.0 Hz), 3.83 (s, 9H), 2.82 (s, 4H), 1.43 (t, 3H, J = 5.0 Hz). 13
C
189
NMR (CDCl3, 101 MHz):
d 153.0, 145.6, 144.0, 137.6, 136.2, 134.9, 190
119.7, 114.6, 111.5, 105.4, 64.6, 60.8, 56.0, 38.4, 37.3, 14.9; MS (m/ 191
z): 333 (M+); ESI-HRMS (m/z): Calcd. for C19H25O5 (M + H)+: 192
333.1702; Found: 333.1741; IR (KBr) (y
max, cmꢀ1): 3345, 2975, 193
2930, 2867, 1592, 1526, 1464, 1425, 1247, 1115, 1003, 869.
194
2.1.6. 2-Ethoxy-5-(3,4,5-trimethoxyphenethyl)phenyl sodium
195
196
phosphate (sit-8)
A solution of phosphorus oxychloride (0.8 mL, 9.0 mmol) in 197
CH2Cl2 (3 mL) was stirred in an ice bath. Compound sit-2 (1 g, 198
3.01 mmol) was dissolved in CH2Cl2 (5 mL) and added. A solution 199
of triethylamine (1.87 mL, 13.5 mmol) in CH2Cl2 (3 mL) was added 200
dropwise after the mixture was stirring for 5 min. TLC analysis 201
indicated the completion of the reaction, then the mixture was 202
quenched by water, extracted with CH2Cl2, dried over Na2SO4 and 203
concentrated. The viscous oil was cooled in an ice bath, neutralized 204
to 8–10 (pH) with 2 mol/L sodium hydroxide and the mixture was 205
stirred for 8 h at 70 8C, then filtered while hot, the filtrate was dried 206
over Na2SO4 and concentrated. Pure white compound sit-8 was 207
obtained in 50% yield by re-crystallization from hot EtOH. mp 208
(CDCl3, 500 MHz):
d 9.82 (s, 1H), 7.51 (d, 1H, J = 5.0 Hz), 7.40–7.46
(m, 6H), 7.06 (d, 1H, J = 10.0 Hz), 6.26 (s, 1H), 5.18 (s, 2H).
3-(Benzyloxy)-4-hydroxybenzaldehyde 10 (1 g, 4.38 mmol)
and potassium carbonate (0.91 g, 6.57 mmol) in DMF (15 mL)
was stirred at 40 8C for 10 min. Ethyl bromide (0.65 mL,
12.0 mmol) was added through the septum after the mixture
was raised to 70 8C. The reaction mixture was then stirred at this
temperature until TLC analysis indicated the completion of the
reaction, then the mixture was quenched by water, extracted with
EtOAc, dried over Na2SO4 and concentrated. Pure white 3-(benzy-
loxy)-4-ethoxybenzaldehyde 6 was obtained by crystallization
(petroleum ether (60–90 8C): EtOAc = 9:1, v/v).
140.3–141.0 8C. 1H NMR (D2O, 500 MHz):
d 7.38 (s, 1H), 6.84 (d, 1H, 209
J = 10.0 Hz), 6.65 (d, 1H, J = 5.0 Hz), 6.54 (s, 2H), 4.04 (q, 2H, 210
J = 5.0 Hz), 3.74 (s, 6H), 3.67 (s, 3H), 2.78–2.81 (m, 4H), 1.33 (t, 3H, 211
Compound 7 (4 g, 7.66 mmol) was dissolved in anhydrous THF
(40 mL), and the mixture was stirred in an ice bath under nitrogen
atmosphere, t-BuOK (1.31 g, 11.7 mmol) was added, then 3-(ben-
zyloxy)-4-ethoxybenzaldehyde 6 (1 g, 3.90 mmol) was dissolved
in anhydrous THF (10 mL) and added through the septum. The
mixture was stirred at room temperature for 4 h. TLC analysis
indicated the completion of the reaction, then the mixture was
quenched by water, extracted with EtOAc, dried over Na2SO4 and
concentrated under reduced pressure. Pure compound sit-11 was
obtained in 78% yield after column chromatography purification
(petroleum ether (60–90 8C): EtOAc = 10:1, v/v). mp 135.8–136 8C.
J = 5.0 Hz). 13C NMR (D2O, 101 MHz):
d
152.1, 146.7, 143.7, 139.0, 212
135.1, 134.7, 121.7, 120.3, 114.2, 105.9, 65.4, 60.7, 55.8, 48.9, 37.4, 213
36.5, 14.1. 31P NMR (D2O, 500 MHz): 3.20(s). MS (m/z): 456 (M+). 214
d
ESI-HRMS (m/z): calculated for C19H23Na2O8P (M+Na)+: 479.0824, 215
found: 479.0813. IR (KBr) (
1589, 1505, 1092, 988.
y
max, cmꢀ1): 3242, 2599, 2239, 1982, 216
217
2.1.7. (Z)-5-(4-Ethoxy-3-nitrostyryl)-1,2,3-trimethoxybenzene
(sit-6)
218
219
A
mixture of 4-hydroxy-3-nitrobenzaldehyde 11 (1 g, 220
1H NMR (CDCl3, 500 MHz):
d
7.49 (d, 2H, J = 5.0 Hz), 7.37–7.41 (m,
6.0 mmol) and potassium carbonate (1.24 g, 9 mmol) in DMF 221
(15 mL) was stirred at 40 8C for 10 min. Ethyl bromide (0.9 mL, 222
12.0 mmol) was added through the septum after the mixture was 223
raised to 70 8C. The reaction mixture was then stirred at this 224
temperature until TLC analysis indicated the completion of the 225
reaction, then the mixture was quenched by water, extracted with 226
EtOAc, dried over Na2SO4 and concentrated. Pure white 4-ethoxy- 227
3-nitrobenzaldehyde 12 was obtained by re-crystallization (pe- 228
troleum ether (60–90 8C): EtOAc = 9:1, v/v). mp 46.4–46.9 8C. 1H 229
2H), 7.32–7.33 (m, 1H), 7.12 (d, 1H, J = 5.0 Hz), 7.05–7.07 (m, 1H),
6.81–6.93 (m, 3H), 6.70 (s, 2H), 5.19 (s, 2H), 4.13 (q, 2H, J = 5.0 Hz),
3.91 (s, 6H), 3.86 (s, 3H), 1.46 (t, 3H, J = 5.0 Hz). 13C NMR (CDCl3,
101 MHz):
d 153.4, 149.2, 148.7, 137.7, 137.3, 133.3, 130.4, 128.5,
127.9, 127.8, 127.3, 126.8, 120.5, 113.7, 112.9, 103.3, 71.5, 64.6,
60.9, 56.1, 14.9; MS (m/z): 421 (M+); ESI-HRMS (m/z): Calcd. for
C26H29O5 (M+H)+: 421.2015, Found: 421.2043; IR (KBr) (ymax
,
cmꢀ1): 2966, 2934, 2835, 2357, 1581, 1508, 1274, 1242, 1130.
NMR (CDCl3, 500 MHz):
1H), 7.21 (d, 1H, J = 10.0 Hz), 4.30 (q, 2H, J = 5.0 Hz), 1.53 (t, 3H, 231
J = 5.0 Hz). Compound (4 g, 7.66 mmol) was dissolved in 232
anhydrous THF (40 mL), and the mixture was stirred in an ice 233
bath under nitrogen atmosphere, n-butyl lithium (2.5 mol/L in 234
hexanes, 2.6 mL) was added, then 4-ethoxy-3-nitrobenzaldehyde 235
12 (1 g, 5.13 mmol) was dissolved in anhydrous THF (10 mL) and 236
added through the septum. The mixture was stirred at room 237
d 9.93 (s, 1H), 8.33 (s, 1H), 8.06–8.08 (m, 230
181
182
183
184
185
186
187
2.1.5. 2-Ethoxy-5-(3,4,5-trimethoxyphenethyl)phenol (sit-2)
A mixture of compound sit-11 (1.0 g, 2.38 mmol) and palladi-
um-carbon (10%, 0.2 g) in MeOH (25 mL) was stirred at room
temperature for 3 h under hydrogen atmosphere. Palladium-
carbon was filtered, the filtrate was dried over Na2SO4 and
concentrated. Compound sit-2 was obtained in 92% yield. mp
7
69.3–69.6 8C. 1H NMR (CDCl3, 500 MHz):
d 6.81 (s, 1H), 6.75 (d, 1H,
Scheme 2. New method for the synthesis of sit-2 and sit-11.
Please cite this article in press as: L. Zhao, et al., Design, synthesis and anti-proliferative effects in tumor cells of new combretastatin A-4
analogs, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.05.003

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L. Zhao et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
temperature for 12 h. TLC analysis indicated the completion of the
reaction, then the mixture was quenched by water, extracted with
EtOAc, dried over Na2SO4 and concentrated under reduced
pressure. Pure compound sit-6 was obtained in 50% yield after
column chromatography purification (petroleum ether (60–90 8C):
EtOAc = 10:1, v/v). mp 101.8–102.4 8C. 1H NMR (CDCl3, 500 MHz):
quenched by saturated NaHCO3 (20 mL), extracted with CH2Cl2,
dried over Na2SO4 and concentrated under reduced pressure. The
crude product was dissolved in MeOH (15 mL) and NaOH solution
(2.5 mL, 2 mol/L) was added. The reaction mixture was stirred at
room temperature for 3 h until the TLC analysis indicated the
completion of the reaction. After cooling, the solvents was
quenched by saturated NaCl solution, extracted with CH2Cl2, dried
over Na2SO4 and concentrated. The residue was purified by column
chromatography (petroleum ether (60–90 8C): EtOAc = 1:1, v/v) to
afford the compound sit-10 in 50% yield. mp 92.1–93.9 8C. 1H NMR
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
d
7.77 (s, 1H), 7.39–7.41 (m, 1H), 6.92 (d, 1H, J = 10.0 Hz), 6.57 (d,
1H, J = 10.0 Hz), 6.43–6.47 (m, 3H), 4.15 (q, 2H, J = 5.0 Hz), 3.85 (s,
3H), 3.72 (s, 6H), 1.46 (t, 3H, J = 5.0 Hz). 13C NMR (CDCl3, 101 MHz):
d
153.2, 151.0, 139.9, 137.7, 134.4, 131.8, 131.2, 129.5, 126.9,
125.8, 114.1, 105.9, 65.5, 61.0, 56.0, 14.5. MS (m/z): 359 (M+). ESI-
HRMS (m/z): calculated for C19H22NO6 (M+H)+: 360.1447, found:
(CDCl3, 500 MHz): d 9.79 (s,1H), 8.38 (s, 1H), 6.77–6.84 (m, 2H),
6.42 (s, 2H), 4.08 (q, 2H, J = 10.0 Hz), 3.84 (d, 9H, J = 5.0 Hz), 3.56 (s,
1H), 2.84 (s, 4H), 2.19 (s, 2H), 1.45 (t, 3H, J = 10.0 Hz). 13C NMR
360.1438. IR (KBr) (y
max, cmꢀ1): 2957, 2835, 1735, 1621, 1577,
1530, 1503, 1458, 1429, 1428, 1412, 1349, 1333, 1164, 1129, 1037,
1005, 968, 930, 856.
(CDCl3, 101 MHz): d 170.4, 153.0, 146.0, 137.8, 136.1, 134.4, 127.6,
123.5, 119.5, 111.1, 100.0, 64.4, 60.9, 56.1, 45.5, 38.7, 37.7, 14.9. MS
(m/z): 388 (M+). ESI-HRMS (m/z): calcd. for C21H29N2O5 (M+H)+,
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
2.1.8. 2-Ethoxy-5-(3,4,5-trimethoxyphenethyl)benzenamine (sit-3)
A solution of compound sit-6 (1.0 g, 2.79 mmol) and palladium-
carbon (10%, 0.2 g) in MeOH/EtOAc (25 mL, 1:1.5, v/v) was stirred
at room temperature for 3 h under hydrogen atmosphere.
Palladium-carbon was filtered off, the filtrate was dried over
Na2SO4 and concentrated. Compound sit-3 was obtained in 90%
389.2076, found: 389.2076. IR (KBr) (y
max, cmꢀ1): 3402, 3271,
2998, 2933, 2835, 1674, 1592, 1540, 1458, 1366, 1334, 1289, 1254,
1235, 1183, 1130, 1079, 1037, 1004, 976, 921, 859.
2.1.11. (Z)-1,2,3-Trimethoxy-5-(4-methoxy-3-nitrostyryl) benzene
(sit-7)
Following the synthetic method from Pettit et al. [11],
compound sit-7 was obtained in 25% yield. 1H NMR (500 MHz,
CDCl3): d8.03 (s, 1H), 7.68 (d, 1H J = 5 Hz), 7.11 (d, 1H, J = 5 Hz), 7.00
(q, 2H, J = 5 Hz), 6.75 (s, 2H), 4.01(s, 3H), 3.94 (s, 6H), 3.90 (s, 3H).
320
321
322
323
324
325
yield. mp 82.9–83.4 8C. 1H NMR (CDCl3, 500 MHz):
d
6.70 (d, 1H,
J = 5.0 Hz), 6.59 (d, 1H, J = 5.0 Hz), 6.51–6.53 (m, 1H), 6.39 (s, 2H),
4.04 (q, 2H, J = 5.0 Hz), 3.83 (s, 9H), 2.75–2.83 (m, 4H), 1.42 (t, 3H,
J = 5.0 Hz). 13C NMR (CDCl3, 101 MHz):
d
153.1, 146.7, 145.9, 137.6,
136.2, 135.2, 134.4, 128.1, 122.7, 119.7, 111.9, 64.3, 60.9, 56.1, 38.5,
37.2. MS (m/z): 331 (M+). ESI-HRMS (m/z): calculated for
2.1.12. 2-Methoxy-5-(3,4,5-trimethoxyphenethyl)benzenamine
(sit-4)
326
327
328
329
330
331
332
333
334
C19H25NaNO4 (M+Na)+, 354.1681, found: 354.1691. IR (KBr) (ymax
,
cmꢀ1): 3434, 3350, 3050, 2986, 2978, 2934, 2837, 1620, 1587,
1517, 1507, 1474, 1455, 1421, 1392, 1325, 1290, 1237, 1226, 1179,
1128, 1048, 1021, 1007, 973, 952, 921, 843.
A solution of compound sit-7 (1.0 g, 2.90 mmol) and palladium-
carbon (10%, 0.2 g) in MeOH/EtOAc (25 mL, 1:1.5, v/v) was stirred
at room temperature for 3 h. Palladium-carbon was filtered off, the
filtrate was dried over Na2SO4 and concentrated. Compound sit-4
was obtained in 90% yield. 1H NMR (500 MHz, CDCl3),
d 6.71 (d, 1H,
J = 10 Hz), 6.58 (d, 1H, J = 5 Hz), 6.55 (d, 1H, J = 10 Hz), 6.39 (s, 2H),
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
2.1.9. 2-Amino-N-(2-ethoxy-5-(3,4,5-trimethoxyphenethyl)phenyl)-
3-hydroxypropanamide (sit-9)
A mixture of compound sit-3 (1 g, 3.02 mmol), Fmoc-
L-serine
3.84 (s, 12H), 2.80 (s, 4H), 1.58(s, 2H).
(1.03 g, 3.15 mmol), DCC (0.65 g, 3.15 mmol), HOBT (0.43 g,
3.15 mmol) in DMF (20 mL) was stirred at room temperature for
5 h under nitrogen atmosphere. TLC analysis indicated the
completion of the reaction, then the mixture was diluted by
EtOAc (10 mL), dried over Na2SO4 and concentrated under reduced
pressure. The crude product was dissolved in CH2Cl2/MeOH (1:1,
10 mL), NaOH solution (2.5 mL, 2 mol/L) was added. The reaction
mixture was stirred at room temperature for 24 h. After cooling,
the solvents was quenched by saturated salt water, extracted with
CH2Cl2, dried over Na2SO4 and concentrated. The residue was
purified by column chromatography (petroleum ether (60–90 8C):
EtOAc = 1:2, v/v) to afford the compound sit-9 in 45% yield. mp
2.1.13. (Z)-2-Methoxy-5-(3,4,5-trimethoxystyryl)benzenamine
(sit-5)
335
336
337
338
339
340
341
342
343
344
A solution of compound sit-7 (1.0 g, 2.90 mmol) and zinc
powder (1.89 g, 29 mmol) in AcOH (30 mL) was stirred at room
temperature for 6 h. The mixture was filtered and the filtrate was
dried over Na2SO4 and concentrated. The residue was purified from
crystallization (petroleum ether (60–90 8C): EtOAc = 9:1, v/v) to
afford compound sit-5 in 73% yield. 1H NMR (500 MHz, CDCl3):
d
6.95 (s, 1H), 6.90 (d, 3H, J = 10 Hz), 6.80 (d, 1H, J = 5 Hz), 6.72 (s,
2H), 3.90 (s, 12H).
162.0–162.5 8C. 1H NMR (CDCl3, 500 MHz):
d
9.92 (s,1H), 8.29 (s,
2.2. Cell growth conditions and anti-proliferative assay
345
1H), 6.82–6.83 (m, 1H), 6.78 (d, 1H, J = 5.0 Hz), 6.40 (s, 2H), 4.07(q,
2H, J = 5.0 Hz), 4.03 (q, 1H, J = 5.0 Hz), 3.88 (q, 1H, J = 5.0 Hz), 3.83
(d, 9H, J = 5.0 Hz), 3.73 (s, 1H), 2.83 (s, 4H), 2.57 (s, 2H), 1.44 (t, 3H,
J = 5.0 Hz). 13C NMR (CDCl3, 101 MHz):
d 171.2, 153.0, 146.2, 137.7,
136.2, 134.3, 127.2, 123.9, 119.7, 111.2, 105.5, 100.0, 64.5, 60.8,
56.6, 56.1, 38.6, 37.6, 29.7, 14.9. MS (m/z): 418 (M+). ESI-HRMS
(m/z): calculated for C22H31N2O6 (M+H)+, 419.2182, found:
419.2170. IR (KBr) (y
max, cmꢀ1): 3366, 3283, 2927, 2012, 1670,
To better characterize drug-induced cytotoxicity of these
compounds (sit-1 to sit-11) in contrast with CA-4, some human
cancer cells like human hepatocellular liver carcinoma cell
(HepG2), human cholangiocarcinoma (QBC939), human breast
cancer cells (SK-BR-3), human colon cancer cell (HCT-8) and
human gastric cancer cell (MKN45) obtained from the Cell Bank of
Chinese Academy of Science were treated. All of these cells were
maintained in RPMI 1640 medium with 10% fetal bovine serum at
37 8C in a humidified atmosphere with 5% CO2. All cells were
seeded into 96-well flat-bottomed culture plates in triplicates
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
1593, 1552, 1507, 1473, 1386, 1329, 1290, 1230, 1185, 1127, 1057,
1011, 972, 890, 871, 854.
295
296
297
298
299
300
2.1.10. 2-Amino-N-(2-ethoxy-5-(3,4,5-
trimethoxyphenethyl)phenyl)acetamide (sit-10)
A mixture of compound sit-3 (1 g, 3.02 mmol), Fmoc-glycine
(0.94 g, 3.15 mmol), BOP (1.40 g, 3.15 mmol) in DMF (20 mL) was
stirred at 60 8C for 2 h under nitrogen atmosphere. TLC analysis
indicated the completion of the reaction, then the mixture was
separately with 10 mg/ml WA or GsA for 44 h. 500 mg/mL 3-(4,5-
dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT)
was added and cells were then incubated for another 4 h. The
IC50 values were calculated through the determination of lactate
dehydrogenase (LDH) in cell culture supernatant using GraphPad
Prism5.0 software.
Please cite this article in press as: L. Zhao, et al., Design, synthesis and anti-proliferative effects in tumor cells of new combretastatin A-4
analogs, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.05.003

G Model
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L. Zhao et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
5
362
2.3. Molecular modeling
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
Energy minimization: Minimum energy conformations of all 11
CA-4 analogs and CA-4 were calculated using the minimize module
of Sybyl-X 2.0 [12]. The force field was calculated with MMFF94 at
˚
an 8 A cutoff for non-bonded interactions, and the atomic point
charges were also calculated using MMFF94. Minimizations were
achieved using the consecutive steepest descent method for the
first 100 steps, conjugate gradient (Powell) and quasi-Newton
(BFGS; named for its originators, and approximates the inverse of
the Hessian matrix) energy minimization steps until the root-
mean-square (RMS) of the gradient became less than
0.005 kcal molꢀ1 A.
˚
Docking calculations: The Surflex-Dock [13] module imple-
mented in the Sybyl program was used for the docking studies. All
CA-4 analogs were docked into a tubulin crystal structure (PDB ID:
3UT5) by an empirical scoring function and a patented search
engine in Surflex-Dock. Protomol, a representation of a ligand
making every potential interaction with the binding site, was
applied to guide molecular docking. Protomols could be estab-
lished by three manners: (1) Automatic: Surflex-Dock finds the
largest cavity in the receptor protein; (2) Ligand: a ligand in the
same coordinate space is used as the receptor; (3) Residues:
residues in the receptor are specified [13]. In this study, the
automatic docking was applied. Other parameters were estab-
lished by default in the software. Surflex-Dock scores (total scores)
were expressed in ꢀlgKd units to represent binding affinity.
388
389
3. Results and discussion
3.1. Chemistry
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
The synthesis of diphenylethane derivates (sit-2, sit-3, sit-6,
sit-8, sit-9, sit-10, sit-11) have been reported by our group before
(Scheme 1) [14]. For the construction of this series of dipheny-
lethane derivatives, the synthesis of 3-(benzyloxy)-4-ethoxyben-
Scheme 3. Synthesis of sit-3, sit-9 and sit-10.
zaldehyde
6 was the main challenge. Firstly, the synthetic
sequence was long. Secondly, the use of expensive, unstable and
toxic metal reducers and the need for slow increase of temperature
from ice bath to room temperatures in the procedures have limited
the product yields. With our continuing interesting in preparing
various CA-4 derivatives, we herein demonstrate a simpler route
for the preparation of various diphenylethane derivates with 3,4-
dihydroxybenzaldehyde 9 using the Wittig reaction as a key step as
illustrated in Scheme 2. 3-(Benzyloxy)-4-hydroxybenzaldehyde 6
was obtained by reaction of commercially material 3,4-dihydrox-
ybenzaldehyde 9 with benzyl bromide [15]. Subsequent alkylation
of the intermediate aldehyde 10 gave 3-(benzyloxy)-4-ethoxy-
benzaldehyde 6.
The modified synthesis of 3-(benzyloxy)-4-hydroxybenzalde-
hyde 6 required only 2 steps compared to 5 steps in the original
method. And the total yield of compound 6 increased from 30% to
63%. A simple, facile, high yield, less cumbersome and environ-
mental friendly synthesis of 3-(benzyloxy)-4-ethoxybenzaldehyde
6 was established.
The aldehyde 6 was reacted with triphenyl(3,4,5-trimethox-
ybenzyl)phosphonium bromine 7 and potassium tert-butoxide
(t-BuOK) to furnish the Wittig product 5-(3-(benzyloxy)-4-
ethoxyphenethyl)-1,2,3-trimethoxybenzene sit-11. Dipheny-
lethene sit-11 was hydrogenated under hydrogen balloon condi-
tions using palladium-carbon as a catalyst to afford the final
product sit-2 in an overall yield of 43%.
The synthesis of the amino derivatives of sit-3, sit-6, sit-9 and
sit-10 are outlined in Scheme 3. 4-Ethoxy-3-nitrobenzaldehyde 12
was obtained from the alkylation of commercial compound
4-hydroxy-3-nitrobenzaldehyde 11. Then sit-6 was prepared from
12 via the Wittig reaction. Sit-3 was easy to synthesis following the 424
general method. DCC and BOP were used to promote the coupling 425
of sit-3 and Fmoc-L-Ser or Fmoc-Gly to produce sit-9 and sit-10. 426
3.2. In vitro anti-proliferative activities
427
The MTT assay was carried out to investigate the anti- 428
proliferative activity of all synthesized CA-4 analogs (sit-1 to 429
sit-11) in human cervix carcinoma (HeLa). CA-4 was used as a 430
positive control in the assay. The IC50 values for sit-1, sit-2, sit-3, 431
sit-4 and sit-8 were 0.22
mmol/L, 0.11 mmol/L, 0.20 mmol/L, 432
0.16 mol/L and 0.57 mol/L, respectively (Table 1). This result 433
m
m
demonstrated that analogs sit-1, sit-2, sit-3 and sit-4 have 434
significant effects in vitro, when compared with the lead compound 435
CA-4 (the IC50 value of CA-4 is 0.40
mmol/L). The anti-proliferative 436
activities of sit-1, sit-2 and sit-3 were also tested in human 437
hepatocellular liver carcinoma cell (HepG2), human cholangio- 438
carcinoma (QBC939), human breast cancer cells (SK-BR-3), human 439
colon cancer cell (HCT-8) and human gastric cancer cell (MKN45). 440
The IC50 value of each analog shows that they all have some effect 441
on each cancer cell in vitro, as shown in Table 2.
442
3.3. Structure–activity relationship study and molecular modeling
443
The double bond in Part A of CA-4 was hydrogenated to afford 444
the final product sit-1. The anti-proliferative effect of sit-1 was 445
2-fold more than that of CA-4, which suggested that the single 446
bond is better than the double bond in Part A for the cytotoxicity in 447
human cancer cells. The IC50 value for sit-4 (a diphenylethane 448
Please cite this article in press as: L. Zhao, et al., Design, synthesis and anti-proliferative effects in tumor cells of new combretastatin A-4
analogs, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.05.003

G Model
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6
L. Zhao et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
Table 1
Structure, purity, yield and in vitro antiproliferative activity of CA-4 analogs on HeLa.
Compound
Structure
Purity (%)
99
Total Yield (%)
32
IC50 value (
0.22
m
mol/L)
Clog P
sit-1
3.07
sit-2
sit-3
sit-4
99
99
96
20/43
0.11
0.20
0.16
3.60
3.46
62
79
sit-5
90
73
No effect
sit-6
sit-7
99
90
50
25
No effect
No effect
4.10
sit-8
sit-9
99
97
50
45
0.57
No effect
1.16
2.01
5.56
sit-10
sit-11
98
99
50
No effect
No effect
22/48
CA-4
98
35
0.40
3.32
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analogs, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.05.003

G Model
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L. Zhao et al. / Chinese Chemical Letters xxx (2015) xxx–xxx
7
Table 2
Cytotoxicity against five cancer cell lines by compounds sit-1, sit-2 and sit-3.
IC50 value (
mmol/L)
HepG2
QBC939
SK-BR-3
HCT-8
MKN45
sit-1
sit-2
sit-3
0.30
0.13
0.19
0.23
0.12
0.24
No effect
0.24
0.85
0.43
0.43
0.89
0.58
1.05
0.28
Fig. 2. Binding conformations of the analogs at the active site of tubulin. (a) Binding model of sit-1 (methoxy derivative, its molecular surface was shown in yellow) and sit-2
(ethoxy derivative, its molecular surface was shown in white). This docking result makes a clear explanation of why the ethoxy is better than the methoxy group for anti-
proliferative effect. (b) and (c) are binding structures of sit-2 (hydroxyl derivative) and sit-3 (amino derivative), respectively. Both hydroxyl derivative and amino derivative
have only one hydrogen bond with the receptor (Asn101).
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
derivative) was 0.16
tive) has no effect in HeLa cells. This result further validated the
above conclusion.
m
mol/L, but sit-5 (a diphenylethene deriva-
be useful for the design of new CA-4 analogs that are structurally 489
related to those used in the current SAR study.
490
The methoxy group in Part B of sit-1 was replaced by an ethoxy
group to afford sit-2. The IC50 value of sit-2 for each human cancer
cells was near half of that of sit-1 in Table 2. It suggested the ethoxy
group is better than the methoxy group for anti-proliferative effect.
The Part C in sit-2 is a hydroxyl group, and in sit-3 is an amino
group. Both compounds have some effect in those six human
cancer cells (Table 2). The anti-proliferative activities of sit-2 and
sit-3 in HepG2, SK-BR-3, HCT-8 are similar. The anti-proliferative
effect of sit-2 was 2-fold more than that of sit-3 in HeLa and
QBC939. There is no significant difference between hydroxyl and
amino groups in Part C for the anti-proliferative effect. But the nitro
group (sit-6 and sit-7) or benzyloxy group (sit-11) can abolish the
anti-proliferative effect (Table 1).
The colchicine binding pocket was used in the study of binding
affinities of our analogs. The total score (a default scoring function
in Sybyl) is an indication of the binding affinity of a ligand to its
receptor. The scores of CA-4 and sit-1 are similar with values of
7.04 and 6.96, respectively. And their anti-proliferative effects are
also similar. The amino acids residues around the Part B in CA-4 are
hydrophobic residues, which suggested the bioactivity can be
increased if the hydrophobicity of Part B in these analogs was
enhanced (Fig. 2). This docking result makes a clear explanation of
why the ethyoxyl is better than the methoxyl group for anti-
proliferative effect. Both the hydroxyl and the amino groups in Part
C can form one hydrogen bond with the receptor (Fig. 2), but the
nitro group or benzyloxy group cannot form any hydrogen bond
with the receptor. It also can explain the difference of the anti-
proliferative effect of analogs sit-2, sit-3, sit-6, sit-7 and sit-11.
As CA-4 does not show in vivo efficacy due to its poor water
solubility, compound sit-8, sit-9 and sit-10 were synthesized as
the pro-drugs of sit-2. The Clog P values (calculated using Sybyl) of
these analogs showed the water-soluble phosphate or amino acid
derivative can improve the water solubility (data shown in
Table 1).
Acknowledgments
491
This work was supported by the National Natural Science 492
Q4
Foundation of China (Nos. 21472126, 21402122) and Shanghai 493
Municipal Natural Science Foundation (No. 12ZR450200).
494
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tested the anti-proliferative effect of each analog, and studied the
structure–activity relationship of those analogs. These results will
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Please cite this article in press as: L. Zhao, et al., Design, synthesis and anti-proliferative effects in tumor cells of new combretastatin A-4
analogs, Chin. Chem. Lett. (2015), http://dx.doi.org/10.1016/j.cclet.2015.05.003